Stilbene synthase genes from grapevine

ABSTRACT

The present invention relates to the gene for stilbene synthase, isolated from plants, and its use for the transformation of vectors, host organisms and plants, as well as to the production of plants which have an increased resistance to pests.

This application is a divisional, of application Ser. No. 08/127,097,filed Sep. 24, 1993 now U.S. Pat. No. 5,500,367; which is a continuationof Ser. No. 07/718,507, filed Jun. 20, 1991, now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates to genes for stilbene synthases, isolatedfrom grapevines, and their use for the transformation of vectors, hostorganisms and plants, as well as to the production of plants which havean increased resistance to pests.

The term stilbenes describes a group of chemical substances which occurin plants and which contain, as the common basic structure, the stilbeneskeleton (trans-1,2-diphenylethylene). This basic skeleton can becomplemented by adding further groups. Two important stilbenes are3,5-dihydroxy-stilbene (pinosylvin) and 3,3',5-trihydroxy-stilbene(resveratrol).

Stilbenes have been found in certain trees (angiosperms, gymnosperms),but also in some herbaceous plants (in species of the families of theMyrtaceae, Vitaceae and Leguminosae). Stilbenes are toxic to pests, inparticular to fungi, bacteria, viruses and insects, and are suitable forfighting these pests. The capability of synthesizing these substancescan be seen as an important defence mechanism of the plants.Unfortunately, only few useful plants are capable of forming stilbenes,or of producing them in such an amount that it makes them sufficientlyresistant to pests.

The use of stilbene synthase genes for producing plants with anincreased pest resistance has already been disclosed in EP-A-0,309,862.This publication specifically describes a stilbene synthase gene frompeanut plants (Arachis hypogea).

There have now been isolated new genes for stilbene synthase ("stilbenesynthase genes") from grapevine, and these genes can be incorporatedinto the genome of plants which produce no stilbenes, or only to aninsufficient extent, by which method the resistance of these plants topests can be increased.

Surprisingly, the new stilbene synthase genes from vine result inconsiderably more favourable pest resistances in plants than thestilbene synthase gene from peanut, which is already known from theprior art.

Stilbene synthase genes are understood as meaning nucleic acids (DNA)which, after they have been transcribed into RNA and translated intoprotein (in a suitable environment) cause the formation of an enzymewhich has the properties of a stilbene synthase (enzymatic synthesis ofstilbene in a suitable environment), these nucleic acids being isolatedfrom their natural environment or being integrated in a vector or beingcontained in a procaryotic or eucaryotic DNA as "foreign" DNA or as"additional" DNA.

Stilbene synthase genes are also understood as meaning those stilbenesynthase genes which contain, at their beginning and/or end, other DNAsequences which do not inhibit the function of the genes, or not to aconsiderable extent, these DNA-sequences which are also termed "geneunits", are formed for example by cutting out using restriction enzymes(for example by cleavage with EcoRI in the case of gene 1 and by partialcleavage in the case of gene 2), since no cleavage sites forconventional restriction enzymes exist exactly at the beginning and atthe end of the gene. At their ends, the stilbene synthase genes, or thegene units, can also carry those DNA sequences which are in each casesuited to their manipulation (for example "linkers").

The stilbene synthase genes (or the gene units) can be present in theform in which they are contained in the plant genome ("genomic" form,including non-stilbene-synthase-encoding and/or non-regulatory sequences(such as introns), or in a form which corresponds to cDNA ("copy" DNA)which can be obtained from mRNA with the aid ofreverse-transcriptase/polymerase (and no longer contains introns). Thestilbene synthase genes can also be in partly or completely synthesizedform. By synthetic genes there are also to be understood genes which arecreated by the novel fusion of portions of natural genes.

SUMMARY OF THE INVENTION

In the stilbene synthase genes (or the gene units) according to theinvention, DNA sections can be replaced by other DNA sections, or DNAs,which act essentially in the same direction.

In the present connection, "foreign" DNA is understood as meaning suchDNA (in particular genes or gene units, or portions thereof) which doesnot naturally occur in a certain procaryotic or eucaryotic genome butwhich is only incorporated into this genome as a result of manipulationby man. "Additional" DNA (in particular genes or gene units, or portionsthereof) will be such DNA which does in fact occur naturally in theparticular procaryotic or eucaryotic genome, but is only incorporatedinto this genome in additional amounts as a result of manipulation byman. One or more specimens of the "foreign" DNA or "additional" DNA canbe incorporated, depending on the demand and the nature of the case inquestion.

Stilbene synthase, which is formed in plants or plant cells underparticipation of the stilbene synthase genes (or the gene units)according to the invention, denotes any enzyme which causes theformation of those vegetable defence substances against pests(phytoalexins) which show a stilbene skeleton.

In the present case, resveratrol is particularly preferred as thestilbene, that is to say, resveratrol synthase is particularly preferredas the stilbene synthase.

As already mentioned, the stilbene synthase genes according to theinvention are those which can be isolated from vine, preferably from(Vitis vinifera and Vitis riparia), with Vitis vinifera being preferred,in particular from Vitis vinifera cv. Optima.

Particularly preferred as stilbene synthase genes according to theinvention are the stilbene synthase genes which are present (as geneunits) in plasmids pVSt1, pVSt2 and pVSt12t3 (which will be described ingreater detail below), as well as the DNA sequences which actessentially in the same direction.

The stilbene synthase genes occurring in grapevines (a number of atleast 5 genes is assumed) display extensive DNA sequence homology. Thestilbene synthase genes contained in plasmid pVSt12t3 are located closeto each other in the plant genome (genetic coupling). All the stilbenesynthase genes encode for resveratrol synthase, the genes differing fromeach other essentially by the quantities of the resveratrol synthaseformed. The stilbene synthase genes, which are not described in detailin the present text, can be determined, isolated and elucidated in thecustomary manner using the known methods of molecular biology because ofthe sequence homology, this being particularly advantageously possiblewith the aid of the gene DNA sequences located on plasmids pVSt12t3,pVSt1 and pVSt2, because of the homologies.

Particularly preferred stilbene synthase genes or gene units accordingto the invention are those contained in plasmid pVSt12t3, as well as theDNA sequences which act in essentially the same direction.

A gene according to the invention, named "gene 1" in the following text,is located in the form of a gene unit, named "gene unit 1" in thefollowing text, on plasmid pVSt12t3 on an EcoRI fragment about 4.9 kb insize. Gene unit 1 can also be isolated from the plasmid using BamHI.HindIII cuts within the gene. Gene unit 1 also exists on plasmid pVSt1and can also be isolated from this plasmid, using the restrictionenzymes mentioned.

A further gene according to the invention, named "gene 2" in thefollowing text, is located in the form of a gene unit, named "gene unit2" in the following text, on plasmid pVSt12t3 on a fragment about 3.4 kbin size, which is obtained by partial digestion with EcoRI. Asp718 cutswithin the gene.

A further functional stilbene synthase gene according to the inventionis obtained by fusing gene unit 2 at the EcoRI site at 3600 bp ofplasmid pVSt12t3 with the EcoRI site at 200 bp of plasmid pVSt12t3. Thefragment of gene unit 2 is here completed by the partial sequence of afurther gene (gene 3), which is contained, in parts, on plasmidpVSt12t3. The two sub-fragments of genes, or gene units, 2 and 3 arealso contained on plasmid pVSt2. The resulting fusion gene is an exampleof a synthetic-gene.

Preferred genes and gene units according to the invention are genes andgene units 1 and 2 and the fusion gene and the corresponding gene unit.Genes and gene units 1 and 2 are particularly preferred.

Escherichia coli strain E. coli Fier 1 pVSt1 contains plasmid pVSt1.Escherichia coli strain E. coli Fier 2 pVSt2 contains plasmid pVSt2.Escherichia coli strain E. coli Fier pVSt12t3 contains plasmid pVSt12t3.These strains were deposited at the Deutsche Sammlung vonMikroorganismen (DSM) German Collection of Microorganisms!, MascheroderWeg 1b, D-3300 Braunschweig, Federal Republic of Germany, in agreementwith the regulations of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure (date of deposit: E. coli Fier 1 pVSt1 and Fier 2 pVSt2: Jun.18, 1990 and E. coli Fier pVSt12t3: Feb. 11, 1991).

Strain E. coli Fier 1 pVSt1 was given Deposit Number DSM 6002, strain E.coli Fier 2 pVSt2 was given Deposit Number DSM 6003 and strain E. coliFier pVSt12t3 was given Deposit Number DSM 6346.

These strains as well as their mutants are likewise part of the presentinvention. The quantities required of plasmids pVSt12t3, pVSt1 andpVSt2, which are deposited in these hosts, can readily be obtained inthe customary manner by multiplying the strains, followed by isolationof the plasmids.

Functionally complete genes, like the stilbene synthase genes accordingto the invention, consist of a regulatory part (in particular promotor)and the structural gene which encodes the protein stilbene synthase.

Both gene portions can be used independently of one another. Forexample, it is possible to arrange downstream of the regulatory portiona different DNA sequence (which deviates from the stilbene synthasegene), which is intended to be expressed after being incorporated intothe plant genome. Since only a few isolated promotors are known whichcan become active in plants or plant cells, the promotors of thestilbene synthase genes, which are likewise parts of the presentinvention, represent valuable tools in the production of transformedplants or plant cells.

Likewise it is possible to arrange a "foreign" regulatory portionupstream of the stilbene synthase structural genes. This might be ofadvantage when, in particular plants, only certain regulatory genes (forexample autologous plant genes) can become active to sufficient extent.The stilbene synthase structural genes therefore represent valuableunits which can be employed on their own, and, as already explained, arelikewise part of the present invention. The stilbene synthase genesaccording to the invention can be separated into the regulatory portionsand the structural genes by the customary method. It is also possiblefor parts of various naturally occurring stilbene synthase genes to becombined to give novel functional "synthetic" genes (for example theabove-described combination of genes 2 and 3). It is preferred to usethe complete natural stilbene synthase genes (or the gene units)according to the invention.

With the aid of the customary methods, it is possible to introduce thestilbene synthase genes (or the gene units) or portions thereof, in one,or more copies (for example tandem arrangement), preferably in one copy,into any procaryotic (preferably bacterial) or eucaryotic (preferablyplant) DNA, as "foreign" or "additional" DNA. For example, genes 1 and 2(or their gene units) can be incorporated together in the way in whichthey are arranged on plasmid pVSt12t3. The recombinant DNA which hasbeen "modified" in this way and which can be used, for example, fortransforming plants or plant cells, and which is contained in plants orplant cells after transformation, is a component of the presentinvention.

The stilbene synthase genes (or the gene units) and/or portions thereof,as well as the recombinant DNA, can be contained in vectors (inparticular plasmids, cosmids or phages), in transformed microorganisms(preferably bacteria, in particular gram-negative bacteria such as E.coli) as well as in transformed plant cells and plants, or in their DNA,as "foreign" or "additional" DNA. Such vectors, transformedmicroorganisms (which can also contain these vectors) as well as thetransformed plant cells and plants and their DNA represent components ofthe present invention.

As already mentioned, one or more copies of the stilbene synthase genes(or the gene units) according to the invention are incorporated into thenatural plant genome (at identical or different sites of the genome), italso being possible for the different genes to be combined with eachother. In the case of plants which are already capable of stilbenesynthesis, the incorporation of one or more stilbene synthase genesaccording to the invention can lead to a considerably improvedresistance behaviour. If appropriate, only the structural genesaccording to the invention are used, and upstream incorporation of aregulatory DNA element isolated from the respective plant may bepossible.

The increased resistance to the transformed plant cells and plantsaccording to the invention is important for agriculture and forestry,for the cultivation of ornamental plants and medicinal plants, and forplant breeding. When plant cells are cultured, for example for obtainingpharmaceutically utilizable substances, it is also advantageous to haveavailable plant cells which have increased resistance to attack bymicrobial pests, in particular fungi.

The present invention therefore also relates to a process for producingtransformed plant cells (including protoplasts) and plants (includingparts of plants, and seeds) with increased resistance to pests, which ischaracterized in that

(a) one or more stilbene synthase genes (or gene units) from grapevineand/or portions of the stilbene synthase genes (or the gene units) fromgrapevine and/or recombinant DNA according to the invention areincorporated into the genome of plant cells (including protoplasts) and,if appropriate,

(b) complete transformed plants are regenerated from the transformedplant cells (including protoplasts) and, if appropriate, propagated,and, if appropriate,

(c) the desired parts of the plants (including seeds) are obtained fromthe resulting transformed plants of the parent generation or of furthergenerations derived therefrom.

Process steps (a), (b) and (c) can be carried out in the customarymanner following known processes and methods.

Transformed plant cells (including protoplasts) and plants (includingparts of plants, and seeds) which contain one or more stilbene synthasegenes (or gene units) from grapevine and/or portions of the stilbenesynthase genes (or of the gene units) from grapevine as "foreign" or"additional" DNA as well as those transformed plant cells and plantswhich can be obtained following the above process, are likewise part ofthe present invention.

Parts of the present invention are also the:

(a) use of the stilbene synthase genes (or of the gene units) fromgrapevine and/or their portions and/or the recombinant DNA according tothe invention and/or the recombinant vectors according to the inventionand/or the transformed microorganisms according to the invention fortransforming plant cells (including protoplasts) and plants (includingparts of plants, and seeds) as well as the

(b) use of the plant cells (including protoplasts) and plants (includingparts of plants, and seeds) transformed according to the invention forproducing propagation material and for producing novel plants andpropagation material thereof, and, generally, the

(c) use of the stilbene synthase genes (or of the gene units) accordingto the invention from grapevine and/or their parts and/or therecombinant DNA according to the invention for combating pests.

A number of various methods is available for incorporating the stilbenesynthase genes or the gene units or their portions into the geneticmaterial of plants or plant cells as "foreign" or "additional" DNA. Thegene transfer can be carried out following the generally customary knownmethods, it being possible for a person skilled in the art to determinethe method which is suitable in each case without difficulty.

A particularly favourable vector which can be employed in many specieswhich is available for transferring foreign DNA into the genes ofdicotyledonous and monocotyledonous plants is the Ti plasmid ofAgrobacterium tumefaciens. The genetic material which encodes stilbenesynthase is introduced into the T-DNA of suitable Ti plasmids (forexample Zambryski et al. 1983) and transferred by infecting the plants,infecting parts of plants or plant tissue, such as, for example, leafdiscs, stalks, hypocotyls, cotyledons, meristems and tissues derivedfrom these such as, for example, secondary embryos and calli, or bycoculturing protoplasts with Agrobacterium tumefaciens.

An alternative is the incubation of purified DNA containing the desiredgene in plant protoplasts (for example Hain et al., 1985; Krens et al.,1982; Paszkowski et al., 1984) in the presence of polycations or calciumsalts and polyethylene glycol.

DNA uptake can also additionally be enhanced by an electrical field(electroporation) (for example Fromm et al. 1986).

The DNA can also be incorporated in a known manner using plant pollen,by "bombarding" pollen with physically accelerated particles which carrythe DNA (cf. EP-A 0,270,356).

The plants are regenerated in a known manner with the aid of suitablemedia (for example Nagy and Maliga 1976).

In a preferred embodiment of the process according to the invention (inaccordance with the method described in EP-A 116,718), the genes or geneunits from plasmids pVSt12t3 or pVSt1 are cloned into a suitableintermediary E. coli vector for example pCV001 or pCV002, (cf. EP-A116,718; Koncz et al. 1986) or, preferably, its derivatives, whichadditionally contain a reporter gene such as, for example, nptII(Herrera-Estrella et al. 1983) or hpt (Van den Elzen et al. 1986).

The plasmid of the above construction is transferred to Agrobacteriumtumefaciens which contains, for example, pGV3850 or its derivatives(Zambryski et al. 1983) using customary methods (for example Van Hauteet al. 1983). Genes 1 and 2 can also be transferred together in thisway. As an alternative, it is possible to clone the stilbene synthasegene unit in a binary vector (for example Koncz and Schell 1986) andtransfer it into a suitable Agrobacterium strain as described above(Koncz and Schell 1986). The resulting Agrobacterium strain whichcontains the stilbene synthase genes or gene units in a form which canbe transferred to plants, is subsequently used for plant transformation.

In a further preferred embodiment, plasmid pVSt12t3 or the isolatedplasmid pVSt1 or the isolated genes or gene units, if appropriatetogether with another plasmid containing a reporter gene for plantcells, for example for kanamycin resistance (for exampleHerrera-Estrella et al. 1983) or a hygromycin resistance (van den Elzen,1986), preferably pLGVneo2103 (Hain et al. 1985), pLGV23neo(Herrera-Estrella 1983), pMON129 (Fraley R. T. et al., Proc. NationalAcad. Sci. U.S.A. 80, 4803 (1983), pAK1003, pAK2004 (Velten J. et al.,EMBO Journ. Vol. 3, 2723 (1984) or pGSSTneo3 (pGSST3) (EP-A-189,707), istransferred to plant protoplasts in a customary manner using direct genetransfer (for example Hain et al. 1985). In this context, the plasmid(s)or the genes or gene units can be present in circular, but preferably inlinear, form. When a plasmid with reporter gene is used,kanamycin-resistant protoplasts are then checked for stilbene synthaseexpression. In the other case (without reporter gene), the resultingcalli are tested for expression of the stilbene synthase gene(s)(screening by customary methods).

Transformed (transgenic) plants or plant cells are produced by the knownmethods, for example by leaf-disc transformation (for example Horsch etal. 1985) by cocultivation of regenerating plant protoplasts or cellcultures with Agrobacterium tumefaciens (for example Marton et al. 1979,Hain et al. 1985) or by direct DNA-transfection. Resulting transformedplants are detected either by selection for reporter gene expression,for example by kanamycin sulphate and in vitro assay for phosphorylationof kanamycin (Reiss et al. 1984; Schreier et al. 1985) or by nopalinsynthase expression (following the method of Aerts et al. 1983) or bystilbene synthase expression using Northern Blot analysis and WesternBlot analysis. Stilbene synthase and the stilbenes can also be detectedin transformed plants in a known manner with the aid of specificantibodies.

Stilbene synthase can also be detected by testing the enzyme activity(Rolfs et al., Plant Cell Reports 1 83-85, 1981).

Culture of the transformed plant cells and regeneration to give completeplants are carried out by the generally customary methods with the aidof the media which are suitable in each case.

The transformed plant cells as well as the transformed plants, all ofwhich contain the stilbene synthase genes (or the gene units) accordingto the invention and which are components of the present invention, showconsiderably increased resistance to pests, in particularphytopathogenic fungi.

The term "plants" in connection with the present invention denotescomplete plants as well as parts of plants such as leaves, seeds,tubers, cuttings etc. "Plant cells" include protoplasts, cell lines,plant calli etc. "Propagation material" denotes plants and plant cellswhich can be used for propagating the transformed plants and plantcells, and is therefore likewise part of the present invention.

The term "DNA sequences which act in essentially the same direction" inthe present connection means that the invention also comprises thosemodifications in which the function of the stilbene synthase genes andportions thereof are not restricted in such a way that stilbene synthaseis no longer formed or that the regulatory gene portion is no longereffective. Modifications in question can be effected by the replacement,the addition and/or the removal of DNA sections, of individual codonsand/or of individual nucleotides.

"Mutants" in connection with the microorganisms which can be usedaccording to the invention denotes those modified microorganisms whichstill show the features which are essential for carrying out theinvention, in particular the plasmids in question.

The plants to which resistance, or increased resistance, to the abovepests can be imparted by incorporating the stilbene synthase genes (orthe gene units) according to the invention (transformation) includevirtually all plants. A particular demand for the provision ofresistance is naturally in crop plants, such as forest plants, forexample firs, spruces, Douglas firs, pines, larches, beeches and oaks,as well as plants which provide food and raw materials, for examplecereals (in particular wheat, rye, barley, oats, millet, rice andmaize), potatoes, Leguminosae such as pulses and, in particular,alfalfa, soya beans, vegetables (in particular cabbage species andtomatoes), fruit (in particular apples, pears, cherries, grapes, citrusfruits, pineapples and bananas), oil palms, tea shrubs, cocoa shrubs andcoffee shrubs, tobacco, sisal and cotton, as well as in medicinal plantssuch as Rauwolfia and Digitalis. Crop plants which may be mentioned asparticularly preferred are potatoes, tomatoes, grapevines and legumes.It is preferred to incorporate the stilbene synthase genes according tothe invention into the genome of plants as "foreign" DNA (that is tosay, in plants with the exception of grapevines).

Pests against which resistances, or increased resistances, can beobtained with the aid of the stilbene synthase gene according to theinvention and which may be mentioned are animal pests such as insects,mites and nematodes, as well as microbial pests such as phytopathogenicfungi, bacteria and viruses. Microbial pests, in particularphytopathogenic fungi, are particularly emphasized.

The harmful insects in particular include insects from the orders ofthe:

Orthoptera, Dermaptera, Isoptera, Thysanoptera, Heteroptera, Homoptera,Lepidoptera, Coleoptera, Hymenoptera and Diptera.

The harmful mites include, in particular: Tarsonemus spp., Panonychusspp. and Tetranychus spp.

The harmful nematodes include, in particular: Pratylenchus spp.,Heterodera spp. and Meloidogyne spp.

The microbial pests include, in particular, the phytopathogenic fungi:

Plasmodiophoromycetes, Oomycetes, Chytridiomycetes, Zygomycetes,Ascomycetes, Basidiomycetes, Deuteromycetes.

The phytopathogenic bacteria include, in particular, thePseudomonadaceae, Rhizobiaceae, Enterobacteriaceae, Corynebacteriaceaeand Streptomycetaceae.

The viral diseases include, in particular, mosaic viruses, stuntingviruses and yellowing viruses.

Some causative organisms of viral, fungal and bacterial diseases whichcome under the generic names listed above may be mentioned as examples,but not by way of limitation:

barley yellow dwarf virus (BYDV), potato virus Y (PVY), cucumber mosaicvirus (CMV), watermelon mosaic virus (WMV), tristeza virus, tobaccomosaic virus (TMV), tobacco necrosis virus (TNV), beet necrotic yellowvein virus (BNYVV) and rhizomania virus.

Xanthomonas species, such as, for example, Xanthomonas campestris pv.oryzae;

Pseudomonas species, such as, for example, Pseudomonas syringae pv.lachrymans;

Erwinia species, such as, for example, Erwinia amylovora; Pythiumspecies, such as, for example, Pythium ultimum; Phytophthora species,such as, for example, Phytophthora infestans;

Pseudoperonospora species, such as, for example, Pseudoperonosporahumuli or Pseudoperonospora cubense;

Plasmopara species, such as, for example, Plasmopara viticola;

Peronospora species, such as, for example, Peronospora pisi or P.brassicae;

Erysiphe species, such as, for example, Erysiphe graminis;

Sphaerotheca species, such as, for example, Sphaerotheca fuliginea;

Podosphaera species, such as, for example, Podosphaera leucotricha;

Venturia species, such as, for example, Venturia inaequalis;

Pyrenophora species, such as, for example, Pyrenophora teres or P.graminea (conidia form: Drechslera, syn: Helminthosporium);

Cochliobolus species, such as, for example, Cochliobolus sativus(conidia form: Drechslera, syn: Helminthosporium);

Uromyces species, such as, for example, Uromyces appendiculatus;

Puccinia species, such as, for example, Puccinia recondita;

Tilletia species, such as, for example, Tilletia caries;

Ustilago species, such as, for example, Ustilago nuda or Ustilagoavenae;

Pellicularia species, such as, for example, Pellicularia sasakii;

Pyricularia species, such as, for example, Pyricularia oryzae;

Fusarium species, such as, for example, Fusarium culmorum;

Botrytis species, such as, for example, Botrytis cinerea;

Septoria species, such as, for example, Septoria nodorum;

Leptosphaeria species, such as, for example, Leptosphaeria nodorum;

Cercospora species, such as, for example, Cercospora canescens;

Alternaria species, such as, for example, Alternaria brassicae and

Pseudocercosporella species, such as, for example, Pseudocercosporellaherpotrichoides. Helminthosporium carbonum may furthermore be mentioned.

The present invention will be illustrated in greater detail with the aidof the following exemplary embodiments:

1. Isolation of the stilbene synthase gene from grapevine

Plants and cell cultures of grapevine (Vitis vinifera, cv. Optima)contain the stilbene synthase genes which cause the formation ofresveratrol synthase (protein size 45,000 D; reaction with specificantiserum).

For the isolation of the stilbene synthase genes, the processes andmethods known in molecular biology were used as are described in detail,for example, in the following handbook: Maniatis, T., Fritsch, E. F.,Sambrook, J.: Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory, Second Edition 1989.

First, a "gene library" for grapevine is established: genomic DNA fromenriched cell nuclei (Bedbrook, J., Plant Molecular Biology Newsletter2, 24, 1981) is cut with the restriction enzyme NdeII in such a way thatDNA fragments of an average length of about 12,000 nucleotide pairs areformed. These fragments are cloned into the BamHI site of lambda phageEMBL4 (Frischauf et al., J. Mol. Biol. 170, 827-842, 1983), and thephages are multiplied in E. coli. The entire phage population containsthe entire genomic DNA of the grapevine cells, cloned in individualfragments, and hence also the stilbene synthases genes (multigenefamily).

The stilbene synthase genes, their mRNA and the cDNA of the stilbenesynthases in each case contain identical nucleic acid sequences sincethey can be derived from each other (gene→mRNA→cDNA). This means thatthe stilbene synthase genes can be identified by specific hybridizationwith stilbene synthase cDNA (Schroder et al. 1988; Melchior and Kindl1990, EP-A-309 862) or with specific oligonucleotides, which can bederived from the DNA sequences indicated in EP-A-309 862. The phageswith the genes are identified by hybridization and then isolated andmultiplied. The genomic DNA from grapevine, which is cloned in thisphage, is further mapped by analysis using various restriction enzymes,and the position of the stilbene synthase genes is determined by furtherhybridization experiments using cDNA sequences or syntheticoligonucleotides. Finally, gene units are cut out of the phage bydigestion with EcoRI or by partial digestion with EcoRI, cloned in theappropriately cut plasmid vector pUC18 (manufacturer: Gibco-BRL GmbH,Eggenstein, Federal Republic of Germany), and multiplied as recombinantplasmids.

For isolating further stilbene synthase genes according to theinvention, DNA sequences which are contained in the genes located onplasmids pVSt12t3, pVSt1 and pVSt2, can be used as probes, because ofsequence homologies. For example, oligonucleotides of the followingsequences can also be used, the figures indicating the position of thenucleotides relative to A of the ATG sequence, which is located at thebeginning of the encoding region:

    ______________________________________                                        (a) 106      TATGC    TGATT   ACTAT  TTCAG                                                 AGTCA    CTAAG   AGCGA  GCACA T 146                              (b) 147      GACTG    AGTTG   AAGAA  GAAGT                                                 TCAAT    CGCAT   ATGTA  A 183                                    (c) 1000     TATGG    TAACA   TGTCT  AGTGC                                                 ATGTG    TCTTG   TTTAT  TTTGG                                                 ATGAG    ATGAG   AAAGA  A 1056                                   ______________________________________                                    

These sequences, or largely homologous sequences, are contained in thestilbene synthase genes according to the invention, so that the lattercan also be characterized by a content of these sequences or by acontent of sequences which are largely homologous to these sequences.

2. Description of plasmids pVSt12t3, pVSt1 and pVSt2 (cf. FIG. 1-FIG. 3)

A) Plasmids pVSt12t3, pVSt1 and pVSt2 consist of two components:

1. Gene units or gene portions:

(a) Plasmid pVSt1:

Gene unit 1, containing the structural gene and the regulator portion ofgene 1 (from grapevine, cv. Optima), is located on a nucleic acid whichcan be Cut out from plasmid pVSt1 as a DNA fragment of (approx) 4900 bpby cleavage with the restriction enzyme EcoRI.

(b) Plasmid pVSt2:

The sequences of gene 2 and gene 3 are located on an EcoRI fragment 3.4kb in size. Circularization of this fragment gives a functionalsynthetic stilbene synthase gene composed of gene portions of genes 2and 3.

(c) Plasmid pVSt12t3:

Plasmid pVSt12t3 contains a genomic Vitis vinifera fragment of approx.12600 bp, flanked by λ-phages DNA in vector pUC18. The complete geneunits 1 and 2 are located on this fragment, which is approx. 12600 bp insize. However, not all of gene 3 is on this fragment. Gene unit 1 can beisolated by EcoRI digestion by a fragment of 4900 bp in length.

Gene unit 2 can be isolated by partial-EcoRI digestion on a 3400 bpfragment.

2. Vector plasmid:

The gene units or gene portions, are cloned in vector pUC18. The size ofthe vector is 2686 nucleotide pairs. It carries the ampicillinresistance gene, that is to say E. coli cells with this plasmid grow innutrient media which contain the antibiotic ampicillin. Ori: term forsequences which are necessary for multiplying the plasmid in E. coli.

Plasmids pVSt1 and pVSt2 carry a gene for ampicillin resistance. Theycan be multiplied in a customary manner in E. coli cells which containpVSt1 or pVSt2 (E. coli Fier 1 and Fier 2, respectively). The sameapplies to E. coli strain Fier pVst12t3.

Preferred nutrient medium for E. coli cells (for example JA221,Nakamura, K., Inouye, M., EMBO J. 1, 771-775, 1982) which contain pVSt1,pVSt2 and pVSt12t3 (E. coli Nurdug 2010):

    ______________________________________                                               Bacto peptone*  10 g                                                          Yeast extract   5 g                                                           NaCl            5 g                                                           Agar            20 g                                                          H.sub.2 O       1 l                                                           pH 7.5                                                                        50 μg/ml of ampicillin are added to the nutrient medium.                   Fermentation: 37° C., aerobic conditions                        ______________________________________                                         (*Bacto is a tradename of DIFCO Lab. Detroit, USA).                      

3. Transformation of tobacco

a) Tobacco shoot culture and isolation of tobacco protoplasts

Nicotiana tabacum (Petit Havanna SR1) is propagated as a sterile shootculture on hormone-free LS medium (Linsmaier and Skoog 1965).Approximately every 6-8 weeks, shoot sections are transferred to freshLS medium. The shoot cultures are maintained in a growth cabinet at24°-26° C. at 12 hours of light (1000-3000 Lux).

For the isolation of leaf protoplasts, about 2 g of leaves (about 3-5 cmin length) are cut into small pieces (0.5 cm×1 cm) using a new razorblade. The leaf material is incubated for 14-16 hours at roomtemperature in 20 ml of enzyme solution consisting of K3 medium (Nagyand Maliga 1976), 0.4 molar sucrose, pH 5.6, 2% of Zellulase R10(Serva), 0.5% of Macerozym R10 (Serva). After this, the protoplasts areseparated from cell debris by filtration over 0.30 mm and 0.1 mm steelsieves. The filtrate is centrifuged for 10 minutes at 100×g. During thiscentrifugation, intact protoplasts float and collect at the top edge ofthe enzyme solution in the form of a band. The pellet consisting of celldebris and the enzyme solution are removed by suction using a glasscapillary. The precleaned protoplasts are made up to 10 ml using freshK3 medium (0.4M of sucrose as an osmotic) and refloated. The wash mediumis removed by suction, and the protoplasts are diluted to 1-2×10⁵ /mlfor culturing or for subsequent infection with agrobacteria (coculture).The protoplast concentration is determined in a haemocytometer.

b) Transformation of regenerating tobacco protoplasts by coculture withAgrobacterium tumefaciens

In what follows, the slightly modified method of Marton et al. 1979 isused. The protoplasts are isolated as described and incubated in K3medium (0.4 molar sucrose, 0.1 mg/l NAA, 0.2 ml in K3 medium (0.4 molarsucrose, 0.1 mg/l NAA, 0.2 mg kinetin) at a density of 1-2×10⁵ /ml for 2days in the dark and for one to two days under weak light (500 lux) at26° C. As soon as the first protoplast divisions take place, 30 μl of anagrobacteria suspension in minimal A (Am) medium (density about 10⁹agrobacteria/ml) are added to 3 ml of regenerating protoplasts.Coculture is effected for 3-4 days at 20° C. in the dark. The tobaccocells are then decanted into 12 ml centrifuge tubes, diluted to 10 mlwith seawater (600 mOsm/kg), and pelleted for 10 minutes at 60×g. Thiswashing process is repeated once or twice so as to remove most of theagrobacteria. The cell suspension is cultured in K3 medium (0.3 molarsucrose) with 1 mg/l NAA (naphthyl-1-acetic acid), 0.2 mg/l kinetin and500 mg/l of the cephalosporin cefotaxim antibiotic at a density of 5×10⁴/ml. The cell suspension is diluted every week using fresh K3 medium,and the osmotic value of the medium is gradually reduced by 0.05 molarsucrose (about 60 mOsm/kg) per week. The selection with kanamycin (100mg/l kanamycin sulphate (Sigma), 660 mg/g active km) is initiated 2-3weeks after coculture in agarose "bead-type culture" (Shillito et al.1983). Kanamycin-resistant colonies can be distinguished from thebackground of retarded colonies 3-4 weeks after the beginning of theselection.

c) Direct transformation of tobacco protoplasts with DNA. Calciumnitrate/PEG transformation

In a Petri dish, about 10⁶ protoplasts in 180 μl of K3 medium arecarefully mixed with 20 μl aqueous DNA solution which contains 0.5 μg/μlplasmid pVSt12t3 or, for example, 0.5 μg/μl plasmid pVSt1 mixed with 0.5μg/μl pLGV neo 2103 (Hain et al. 1985). 200 μl fusion solution (0.1molar calcium nitrate, 0.45M of mannitol, 25% of polyethylene glycol(PEG 6000), pH 9) are subsequently added carefully. After 15 minutes, 5ml of wash solution (0.275M of calcium nitrate, pH 6) are added and,after a further 5 minutes, the protoplasts are transferred to acentrifuge tube and pelleted at 60×g. The pellet is taken up in a smallamount of K3 medium and cultured as described in the section below.Alternatively, the protoplasts can be transformed as described by Hainet al. 1985.

The transformation can also be carried out without the addition of the0.5 μg/μl pLGVneo2103. Since no reporter gene is employed in this case,the resulting calli can be tested for the presence of the stilbenesynthase gene unit with the aid of a dot-blot hybridization.Hybridization probes which can be used are internal EcoRI fragments frompVSt1 or pVSt2. Other detection methods such as antibody assay ordetection of an increased resistance to fungi can, of course, also beemployed.

d) Culture of the protoplasts incubated together with DNA, and selectionof kanamycin-resistant calli:

For the culture described below and for the selection ofkanamycin-resistant colonies, a modified "bead-type culture" technique(Shillito et al. 1983) is used. One week after the protoplasts have beentreated with DNA (cf. c), 3 ml of the cell suspension are mixed with 3ml of K3 medium (0.3M of sucrose+hormones; 1.2% of (Seaplaque) LMTagarose (low-melting agarose, Marine Colloids) in 5 cm Petri dishes. Forthis purpose, dry agarose is autoclaved, K3 medium is added, and themixture is briefly boiled up in a microwave oven. After the agarose hassolidified, the agarose discs ("beads") together with the embeddedtobacco microcalli are transferred into 10 cm Petri dishes for furtherculture and selection, and batches of 10 ml of K3 medium (0.3M ofsucrose, 1 mg/l NAA, 0.2 mg/l kinetin) and 100 mg/l kanamycin sulphate(Sigma) are added. The liquid medium is changed every week. During thisprocess, the osmotic value of the medium is gradually lowered.

The exchange medium (K3 +km) is reduced every week by 0.05 molar sucrose(about 60 mOsm).

Selection diagram for kanamycin-resistant tobacco colonies after DNAtransformation:

    ______________________________________                                                                                   Sucrose in                             0.4M    0.3M   0.25M 0.20M 0.15M 0.10M the liquid                         U   E S                        K           medium                             ______________________________________                                        .   1       2      3     4     5     6     Weeks after                                                                   DNA uptake                         (K3 medium, 1 mg of NAA, 0.2 mg of kinetin)                                   ______________________________________                                         U = DNA uptake                                                                E = Embedding in agarose                                                      S = Selection with kanamycin (100 mg/l of kanamycin sulphate)                 K = Kanamycinresistant colonies can be distinguished clearly from the         background                                                               

e) Regeneration of kanamycin-resistant plants

As soon as the kanamycin-resistant colonies have reached a diameter ofabout 0.5 cm, half of them are placed on regeneration medium (LS medium,2% of sucrose, 0.5 mg/l benzylaminopurine BAP), and the cultures aremaintained in a growth cabinet at 24° C. with 12 hours of light(3000-5000 lux). The other half is propagated as a callus culture on LSmedium containing 1 mg/l NAA, 0.2 mg/l kinetin, 0.1 mg/l BAP and 100mg/l kanamycin sulphate. When the regenerated shoots have a size ofabout 1 cm, they are excised and placed on 1/2 LS medium (1% of sucrose,0.8% of agar) without growth regulators, for rooting. The shoots arerooted on 1/2 MS medium containing 100 mg/l kanamycin sulphate, andlater transplanted into soil.

f) Transformation of leaf discs by Agrobacterium tumefaciens

For the transformation of leaf discs (Horsch et al. 1985), leaves, about2-3 cm in length, of sterile shoot cultures, are punched into discs ofdiameter 1 cm, and the discs are incubated for about 5 minutes with asuspension of appropriate agrobacteria (about 10g/ml) (cf. b) in the Ammedium, see below). The infected leaf portions are maintained on MSmedium (see below) without hormones for 3-4 days at about 24° C. Duringthis time, Agrobacterium covers the leaf portions with growth. The leafportions are subsequently washed in MS medium (0.5 mg/ml BAP, 0.1 mg/mlNAA) and placed on the same medium (0.8% of agar) containing 500 μg/mlcefotaximand 100 μg/ml kanamycin sulphate (Sigma). The medium should berenewed after two weeks. Transformed shoots become visible after afurther 2-3 weeks. The regeneration of shoots should also be carried outin parallel without selection pressure. The regenerated shoots must thenbe tested for transformation, using biological tests for example fornopalin synthase or stilbene synthase activity. 1-10% of transformedshoots are obtained in this manner.

Biochemical method for detecting transformation

Detection of nopalin in plant tissues

Nopalin is detected as follows, as described by Otten and Schilperoort(1978) and Aerts et al. (1979). In an Eppendorf container, 50 mg ofplant material (callus or leaf portions) are incubated overnight in LSmedium containing 0.1M of arginine at room temperature. The plantmaterial is then dabbed with absorbent paper, homogenized in a newEppendorf centrifuge container using a glass rod, and the homogenate iscentrifuged for 2 minutes in an Eppendorf centrifuge. 2 μl of thesupernatant are applied to a sheet of paper (20×40 cm) which is suitablefor electrophoresis (Whatman 3 MM paper) in the form of small spots andthen dried. The sheet of paper is saturated with the mobile phase (5% offormic acid, 15% of acetic acid, 80% of H₂ O, pH 1.8), andelectrophoresis is carried out for 45 minutes at 400 V. Nopalin migratesto the cathode. The sheet of paper is then dried in a stream of hot airand pulled through phenanthrenequinone stain (equal volumes of 0.02%strength phenanthrenequinone in ethanol and 10% strength NaOH in 60%strength ethanol) in the direction of movement. The dried sheet of paperis viewed under long-wave UV light, and photographs are taken. Thereagent stains arginine and arginine derivatives fluorescent yellow.

Neomycin phosphotransferase (NPT II) enzyme assay

NPT II activity in plant tissue is detected by in situ phosphorylationof kanamycin as described by Reiβ et al. (1984) and modified by Schreieret al. (1985), as follows. 50 mg of plant tissue are homogenized in 50μl of extraction buffer (10% of glycerine, 5% of 2-mercaptoethanol, 0.1%of SDS, 0.025% of bromphenol blue, 62.5 mM of tris pH 6.8) with theaddition of glass powder, on ice, and the homogenate is centrifuged for10 minutes in an Eppendorf centrifuge at 4° C. 50 μl of the supernatantare applied to a native polyacrylamide gel (145×110×1.2 mm; separatinggel: 10% of acrylamide, 0.33% of bisacrylamide, 0.375M of tris pH 8.8,collecting gel: 5% of acrylamide, 0.165% of bisacrylamide, 0.125M oftris pH 6.8) and the gel is subjected to electrophoresis overnight at 4°C. and 60 V. As soon as the Bromphenol Blue marker moves out of the gel,the gel is washed twice with distilled water for 10 minutes and oncewith reaction buffer (67 mM tris maleate, pH 7.1, 42 mM of MgCl₂, 400 mMof ammonium chloride) for 30 minutes. The gel is placed on a glass plateof equal size and covered with 40 ml of 1% strength agarose in reactionbuffer containing the substrates kanamycin sulphate (20 μg/ml) and20-200 μCi ³² P ATP (Amersham). The sandwich gel is incubated for 30minutes at room temperature, and a sheet of phosphocellulose paper P81(Whatman) is then placed on the agarose. Four layers of filter paper 3MM (Whatman) and some paper towels are then placed on top of this. Thetransfer of in situ phosphorylated radioactive kanamycin phosphate tothe P81 paper is stopped after 3-4 hours. The P81 paper incubated for 30minutes in a solution of Proteinase K and 1% strength sodium dodecylsulphate (SDS) at 60° C. and then washed 3-4 times in 250 ml of 10 mMphosphate buffer pH 7.5 at 80° C., dried, and autoradiographed for 1-12hours at -70° C. (XAR5 film, Kodak).

Transformation of Solanum tuberosum (potato)

The transformation was transformed precisely in the manner given inEP-A-0,242,246, pages 14 to 15, the agrobacteria containing Ti plasmidscarrying the stilbene synthesis gene or the stilbene synthase genes.

All percentages in the above examples relate to per cent by weightunless indicated otherwise.

In the plant cells and plants (tobacco) obtained in accordance with theabove examples, the presence of the stilbene synthase genes wasconfirmed by Southern Blot analysis. The expression of the stilbenesynthase genes was detected by Northern Blot analysis, and stilbenesynthase and stilbenes with the aid of specific antibodies. Transformedand non-transformed plants (for comparison) were sprayed with a sporesuspension of Botrytis cinera, and, after 1 week, the plants were scoredfor fungal disease. The transformed plants showed an increasedresistance to fungal disease (compared with the non-transformedcomparison plants).

In what follows, some of the media employed in the transformation ofplants or plant cells are described:

    ______________________________________                                        Am medium                                                                     ______________________________________                                               3.5 g      of K.sub.2 HPO.sub.4                                               1.5 g      of KH.sub.2 PO.sub.4                                               0.5 g      of Na.sub.3 citrate                                                0.1 g      of MgSO.sub.4 × 7H.sub.2 O                                   1 g        of (NH.sub.4).sub.2 SO.sub.4                                       2 g        of glucose                                                                    to 1 l                                                      ______________________________________                                    

Medium for sterile shoot culture of tobacco

Macro elements 1/2 of the concentration of the MS salts Micro elements1/2 of the concentration of the MS salts Fe EDTA Murashige and Skoog(MS)

    ______________________________________                                        Myo-inositol            100    mg/l                                           Sucrose                 30     g/l                                            Agar                    8      g/l                                            Vitamins     Ca panthotenate                                                                              1      mg/l                                                    Biotin         10     mg/l                                                    Nicotinic acid 1      mg/l                                                    Pyridoxine     1      mg/l                                                    Thiamine       1      mg/l                                       pH 5.7 before autoclaving                                                     ______________________________________                                    

K3 medium

For culturing Nicotiana tabacum petit Havana SR1, Nicotiana tabacumWisconsin 38, and Nicotiana plumaginifolia protoplasts (Nagy and Maliga,1976)

    ______________________________________                                        Macro elements                                                                            NH.sub.4 NO.sub.3                                                                            250    mg/l                                                    KNO.sub.3      2500   mg/1                                                    CaCl.sub.2 × 2H.sub.2 O                                                                900    mg/l                                                    MgSO.sub.4 × 7H.sub.2 O                                                                250    mg/l                                                    NaH.sub.2 PO.sub.4 × 1H.sub.2 O                                                        150    mg/l                                                    (NH.sub.4).sub.2 SO.sub.4                                                                    134    mg/l                                                    CaHPO.sub.4 × 1H.sub.2 O                                                               50     mg/l                                        Micro elements                                                                            H.sub.3 BO.sub.3                                                                             3      mg/l                                                    MnSO.sub.4 × 1H.sub.2 O                                                                10     mg/l                                                    ZnSO.sub.4 × 4H.sub.2 O                                                                2      mg/l                                                    KI             0.75   mg/l                                                    Na.sub.2 MoO.sub.4 × 2H.sub.2 O                                                        0.25   mg/l                                                    CuSO.sub.4 × 5H.sub.2 O                                                                0.025  mg/l                                                    CoCl.sub.2 × 6H.sub.2 O                                                                0.025  mg/l                                        Fe-EDTA     Na.sub.2 EDTA  37.2   mg/l                                                    FeSO.sub.4 × 7H.sub.2 O                                                                27.8   mg/l                                        Inositol                   100    mg/l                                        Sucrose                    137    g/l                                                                           (=0.4 M)                                    Xylose                     250    mg/l                                        Vitamins    Nicotinic acid 1      mg/l                                                    Pyridoxine     1      mg/l                                                    Thiamine       10     mg/l                                        Hormones    NAA            1.0    mg/l                                                    Kinetin        0.2    mg/l                                        pH 5.6                                                                        Filter-sterilization                                                          ______________________________________                                    

Linsmaier and Skoog medium (Linsmaier and Skoog 1965)

For culturing regenerating protoplasts and for tissue culture of tobaccotumours and callus. Linsmaier and Skoog (LS) medium is Murashige andSkoog medium (Murashige and Skoog, 1962) with the followingmodifications:

The thiamine weighed in is more concentrated 0.4 mg/l instead of 0.1mg/l;

No glycine, pyridoxine and nicotinic acid.

    ______________________________________                                        Macro elements                                                                            NH.sub.4 NO.sub.3                                                                            1650   mg/l                                                    KNO.sub.3      1900   mg/1                                                    CaCl.sub.2 × 2H.sub.2 O                                                                440    mg/l                                                    MgSO.sub.4 × 7H.sub.2 O                                                                370    mg/l                                                    KH.sub.2 PO.sub.4                                                                            170    mg/l                                        Micro elements                                                                            H.sub.3 BO.sub.3                                                                             6.2    mg/l                                                    MnSO.sub.4 × 1H.sub.2 O                                                                22.3   mg/l                                                    ZnSO.sub.4 × 4H.sub.2 O                                                                8.6    mg/l                                                    KI             0.83   mg/l                                                    Na.sub.2 MoO.sub.4 × 2H.sub.2 O                                                        0.25   mg/l                                                    CuSO.sub.4 × 5H.sub.2 O                                                                0.025  mg/l                                                    CoCl.sub.2 × 6H.sub.2 O                                                                0.025  mg/l                                        Fe-EDTA     Na.sub.2 EDTA  37.2   mg/l                                                    FeSO.sub.4 × 7H.sub.2 O                                                                27.8   mg/l                                        Inositol                   100    mg/l                                        Sucrose                    30     g/l                                         Agar                       8      g/l                                         Vitamins    Thiamine       0.4    mg/l                                        Hormones:   NAA            1      mg/l                                                    Kinetin        0.2    mg/l                                        pH 5.7 before autoclaving                                                     ______________________________________                                    

The following literature on the transformation of plants or plant cellsmay be cited:

Aerts M., Jacobs M., Hernalsteens J. P., Van Montagu M., Schell J.(1983) Induction and in vitro culture of Arabidopsis thaliana crown galltumours. Plant Sci Lett. 17: 43-50

Fraley R. T., Rogers S. G., Horsch R. B., Sanders P. R., Flick J. S.,Adams S. P., Bittner M. L., Brand L. A., Fink C. L., Fry J. S., FallupiG. R., Goldberg S. B., Hoffmann N. L., Woo S. C. (1983). Expression ofbacterial genes in plant cells. Proc. Natl. Acad. Sci. U.S.A.80:4803-4807.

Fromm M. E., Taylor L. P., Walbot V. (1986) Stable transformation ofmaize after gene transfer by electroporation. Nature 319: 791-793

Hain, R., Stabel, P., Czernilofsky, A. P., Steinbiβ, H. H.,Herrera-Estrella, L., Schell, J. (1985) Uptake, integration, expressionand genetic transmission of a selectable chimeric gene by plantprotoplasts. Molec Gen Genet. 199: 161-168

Hain R., Bieseler B., Kindl H., Schroder G., Stocker R. (1990)Expression of a stilbene synthase gene in Nicotiana tabacum results insynthesis of the phytoalexin resveratrol. Plant Mol Biol. 15:325-336.

Hernalsteens J. P., Thia-Tong L., Schell J., Van Montagu M. (1984) AnAgrobacterium-transformed Cell culture from the monocot Asparagusofficinalis. EMBO J 3:3039-3041

Herrera-Estrella L., De Block M., Messens E., Hernalsteens J. P., vanMontagu M., Schell J. (1983) EMBO J. 2: 987-995.

Horsch R. B., Fry J. E., Hoffmann N. L., Eichholtz D., Rogers S. G.,Fraley R. T. (1985) A simple and general method for transferring genesinto plants. Science 277:1229-1231

Krens F. H., Molendijk L., Wullems G. J., Schilperoort R. A. (1982) invitro transformation of plant protoplasts with Ti-plasmid DNA. Nature296: 72-74

Koncz C., Schell J. (1986) The promoter of T_(L) -DNA gene 5 controlsthe tissue-specific expression of chimaeric genes carried by a novaltype of Agrobacterium linary vector. Mol. Gen. Genet. (1986) 204:338-396

Linsmaier D. M., Skoog F. (1965) Organic growth factor requirements oftobacco tissue cultures. Physiol Plant 18: 100-127

Marton L., Wullems G. J., Molendijk L., Schilperoort P. R. (1979) Invitro transformation of cultured cells from Nicotiana tabacum byAgrobacterium tumefaciens. Nature 277: 1229-131

Melchior F., Kindl H. (1990) Grapevine stilbene synthase cDNA onlyslightly differing from chalcone synthase cDNA is expressed inEscherichia coli into a catalytically active enzyme FEBS 268:17-20

Nagy J. I., Maliga P. (1976) Callus induction and plant regenerationfrom mesophyll protoplasts of Nicotiana sylvestris. Z. Pflanzenphysiol78: 453-455

Otten L. A. B. M., Schilperoort R. A. (1978) A rapid microscale methodfor the detection of Lysopin and Nopalin dehydrogenase activities.Biochim biophys acta 527: 497-500

Paszkowski J., Shillito R. D., Saul M., Mandak V., Hohn T., Hohn B.,Potrykus I. (1984) Direct gene transfer to plants. EMBO J 3: 2717-2722

Rolf, C. H., Fritzemeier K. H. and Kindl H. (1981) Cultured cells ofArachis hypogaea susceptible to induction of stilbene synthase(resveratrol forming) Plant Cell. Rep. 1:83-85

Schroder, G., Brown J. W. S. and Schroder, J. (1988) Molecular analysisof resveratrol synthase: cDNA, genomic clones and relationship withchalconsynthase. Eur. J. Biochem. 172, 161-169

Shillito R. D., Paszkowski J. Potrykus I. (1983) Agarose plating andBead type culture technique enable and stimulate development ofprotoplast-derived colonies in an number of plant species. Pl Cell Rep2: 244-247

Van den Elzen P. J. M., Townsend J., Lee K. Y., Bedbrook J. R. (1985) Achimaeric resistance gen as a selectable marker in plant cells. PlantMol. Biol. 5, 299-302.

Velten J., Velten L., Hain R., Schell J. (1984) Isolation of a dualplant promotor fragment from the Ti Plasmid of Agrobacteriumtumefaciens. EMBO J 12: 2723-2730

Van Haute E., Joos H., Maes M., Warren G., Van Montagu M., Schell J.(1983) Intergenic transfer and excharge recombination of restrictionfragments clones in pBR 322: a novel strategy for the reversed geneticsof Ti plasmids of/Agrobacterium tumefacines. EMBO J 2: 411-418.

Zambryski P., Joos H., Genetello C., van Montagu M., Schell J. (1983)Ti-plasmid vector for the introduction of DNA into plant cells withoutaltering their normal regeneration capacity, EMBO J 12: 2143-2150.

Reiss, B., Sprengel, Will H., and Schaller H.(1984) A new sensitivemethod for qualitative and quantitative assay of neomycinphosphotransferase in crude cell tracts, GENE 1081: 211-217

Schreier P. H., Seftor E. A., Schell J. and Bohnert H. J. (1985) The useof nuclear-encoded sequences to direct the light-regulated synthesis andtransport of a foreingn protein into plant chloroplasts, EMBO J Vol. 4,No. 1: 25-32

The following published patent applications can furthermore be listed:

EP-A-116,718 EP-A-126,546

EP-A-159,418 EP-A-164,597

EP-A-120,515 EP-A-175,966

EP-A-120,516 WO 84/02913

EP-A-172,112 WO 84/02919

EP-A-140,556 WO 84/02920

EP-A-174,166 WO 83/01176

EP-A-122,791

EXPLANATIONS OF FIG. 1-FIG. 4

FIG. 1 represents plasmid pVSt12t3. The stilbene synthase gene units 1and 2 are located on a Vitis DNA fragment, approx. 12,600 bp in length,which, flanked in pVSt12t3 by λ-phage DNA, had been cloned into the SmaIsite of pUC18. Only terminal sequences of gene 3 are located onpVSt12t3. 1 in each case denotes the beginning of the encoding region,and 2 the end of the encoding region, of gene 1, gene 2 and gene 3. Inaddition, the arrows indicate how the genes are orientated on thefragment. The emboldened areas represent flanking phage DNA.

FIG. 2 represents plasmid pVSt1. Gene 1 is located on the EcoRIfragment, approx. 4.9 kb in size. It can also be isolated by doubledigestion with BamHI and EcoRI. HindIII cuts within the gene. The arrowindicates how the gene is orientated on the plasmid.

FIG. 3 represents plasmid pVSt2. This plasmid contains an EcoRI fragmentof pVSt12t3, 3.4 kb in size, which contains the terminal areas of gene 3and the proximal areas of gene 2. Circularization of this fragmentresults in a functional stilbene synthase gene. This gene is a syntheticgene of gene 2 and gene 3.

FIG. 4 represents encoding (exons/emboldened) and non-encoding areas(introns) of gene 1 and gene 2. The position of the translationinitiation codon (ATG) is at approximately 11,000 (see FIG. 1) in thecase of gene 1 and in position 2,550 in the case of gene 2. Gene 1 has alength of approximately 1,530 bp and ends in position 9466. Gene 2 isaprox. 1,300 bp in length and ends in position 3861. The differentlength of the two genes is the result of introns of different lengths.

In FIGS. 1-4, the symbols denote:

E: EcoRI

B: BamHI

H: HindIII

PL: Polylinker from plasmid pUC18

A: Asp718

ATG: Translation initiation codon

S: SmaI

The increased resistance of plants which have been transformed accordingto the invention will be illustrated with the aid of the followingexample:

DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLE A

To test for an increased resistance to plant diseases, the plants wereinoculated with a pathogen, and the degree of infection was used as theparameter. The test pathogen used was Botrytis cinerea Pers.

The tobacco plants were preculturedbytissue culture and subsequentlypotted in the greenhouse in standard soil (manufactured by Balster) inpots (d=11 cm), and raised in the greenhouse at 23° C. and 70-80 %relative atmospheric humidity until the tests were started. They weresupplied with water and fertilizer as required. For inoculation, theleaves of the plants (3-4 weeks after transferral to the greenhouse)were sprayed to runoff point with a spore suspension of the pathogen.The plants were then incubated at 100 % relative atmospheric humidityand 10°-20° C. After 4-8 days, the health of the plants was determinedas a percentage, based on the infected leaf area.

As can be seen from the tables, transformed tobacco plants, in which astilbene synthase gene according to the invention was introduced, wereinfected with B. cinerea to a lesser degree than those of thenon-transformed wild type SR1, and therefore showed an increasedresistance to fungi in comparison with the non-transformed controlplants.

                  TABLE                                                           ______________________________________                                        Effect of the resveratrol gene on infection of the                            tobacco plants with Botrytis cinerea.                                         % infected leaf area on leaf No.                                              1          2      3      4    5   6   x    Reduction*                         ______________________________________                                        Wild type                                                                             15.0   16.0   16.5 11.5 8.6 2.8 11.7                                  (control)                                                                     Trans-  6.4    8.6    8.8  2.9  1.7 0.9 4.9  58%                              formed                                                                        tobacco 1                                                                     Trans-  10.8   8.1    3.7  3.6  3.0 0.8 5.0  57%                              formed                                                                        tobacco 2                                                                     ______________________________________                                         *Reduction calculated using Abbott's formula                                  x = Averaqe                                                              

We claim:
 1. A transgenic plant cell comprising within its genome a DNAfragment selected from the group consisting of:(i) a restriction enzymedigestion fragment of genomic grapevine (Vitis) DNA, said fragmentencoding a resveratrol synthase, being approximately 4900 base pairs insize and being obtainable from plasmid pVSt12t3 by cleavage with EcoRI;(ii) a restriction enzyme digestion fragment of genomic grapevine(Vitis) DNA, said fragment encoding a resveratrol synthase, beingapproximately 3400 base pairs in size and being obtainable from plasmidpVSt12t3 by partial cleavage with EcoRI; (iii) a restriction enzymedigestion fragment of genomic grapevine (Vitis) DNA, said fragmentencoding a resveratrol synthase, being approximately 12600 base pairs insize and being obtainable from plasmid pVSt12t3 by cleavage with SmaI;(iv) a cDNA encoding said resveratrol synthase of (i), (ii) or (iii);(v) a DNA encoding said reveratrol synthase of (i), (ii) or (iii); and(vi) a synthetic DNA comprising (i), (ii), (iii), (iv) or (v);whereinsaid DNA fragment is expressed when said transgenic plant cell isexposed to a pest, said transgenic plant cell exhibiting increasedresistance to said pest as compared to a non-transgenic plant cell ofthe same cell type and plant species exposed to the same pest under thesame conditions, and said increased resistance to said pest being aresult of the expression of said DNA fragment.
 2. A transgenic plantcell according to claim 1, comprising within its genome a restrictionenzyme digestion fragment of genomic grapevine (Vitis) DNA, saidfragment encoding a resveratrol synthase, being approximately 4900 basepairs in size and being obtainable from plasmid pVSt12t3 by cleavagewith EcoRI.
 3. A transgenic plant cell according to claim 1, comprisingwithin its genome a restriction enzyme digestion fragment of genomicgrapevine (Vitis) DNA, said fragment encoding a resveratrol synthase,being approximately 3400 base pairs in size and being obtainable fromplasmid pVSt12t3 by partial cleavage with EcoRI.
 4. A transgenic plantcell according to claim 1, comprising within its genome a restrictionenzyme digestion fragment of genomic grapevine (Vitis) DNA, saidfragment encoding a resveratrol synthase, being approximately 12600 basepairs in size and being obtainable from plasmid pVSt12t3 by cleavagewith SmaI.
 5. A transgenic whole plant comprising within its genome aDNA fragment selected from the group consisting of:(i) a restrictionenzyme digestion fragment of genomic grapevine (Vitis) DNA, saidfragment encoding a resveratrol synthase, being approximately 4900 basepairs in size and being obtainable from plasmid pVSt12t3 by cleavagewith EcoRI; (ii) a restriction enzyme digestion fragment of genomicgrapevine (Vitis) DNA, said fragment encoding a resveratrol synthase,being approximately 3400 base pairs in size and being obtainable fromplasmid pVSt12t3 by partial cleavage with EcoRI; (iii) a restrictionenzyme digestion fragment of genomic grapevine (Vitis) DNA, saidfragment encoding a resveratrol synthase, being approximately 12600 basepairs in size and being obtainable from plasmid pVSt12t3 by cleavagewith SmaI; (iv) a cDNA encoding said resveratrol synthase of (i), (ii)or (iii); (v) a DNA encoding said resveratrol synthase of (i), (ii) or(iii); and (vi) a synthetic DNA comprising (i), (ii), (iii), (iv) or(v);wherein said DNA fragment is expressed when said transgenic wholeplant is exposed to a pest, said transgenic whole plant exhibitingincreased resistance to said pest as compared to a non-transgenic wholeplant of the same plant species exposed to the same pest under the sameconditions, and said increased resistance to said pest being a result ofthe expression of said DNA fragment.
 6. A transgenic whole plantaccording to claim 5, comprising within its genome a restriction enzymedigestion fragment of genomic grapevine (Vitis) DNA, said fragmentencoding a resveratrol synthase, being approximately 4900 base pairs insize and being obtainable from plasmid pVSt12t3 by cleavage with EcoRI.7. A transgenic whole plant according to claim 5, comprising within itsgenome a restriction enzyme digestion fragment of genomic grapevine(Vitis) DNA, said fragment encoding a resveratrol synthase, beingapproximately 3400 base pairs in size and being obtainable from plasmidpVSt12t3 by partial cleavage with EcoRI.
 8. A transgenic whole plantaccording to claim 5, comprising within its genome a restriction enzymedigestion fragment of genomic grapevine (Vitis) DNA, said fragmentencoding a resveratrol synthase, being approximately 12600 base pairs insize and being obtainable from plasmid pVSt12t3 by cleavage with SmaI.9. A transgenic plant part comprising within its genome a DNA fragmentselected from the group consisting of:(i) a restriction enzyme digestionfragment of genomic grapevine (Vitis) DNA, said fragment encoding aresveratrol synthase, being approximately 4900 base pairs in size andbeing obtainable from plasmid pVSt12t3 by cleavage with EcoRI; (ii) arestriction enzyme digestion fragment of genomic grapevine (Vitis) DNA,said fragment encoding a resveratrol synthase, being approximately 3400base pairs in size and being obtainable from plasmid pVSt12t3 by partialcleavage with EcoRI; (iii) a restriction enzyme digestion fragment ofgenomic grapevine (Vitis) DNA, said fragment encoding a resveratrolsynthase, being approximately 12600 base pairs in size and beingobtainable from plasmid pVSt12t3 by cleavage with SmaI; (iv) a cDNAencoding said resveratrol synthase of (i), (ii) or (iii); (v) a DNAencoding said resveratrol synthase of (i), (ii) or (iii); and (vi) asynthetic DNA comprising (i), (ii), (iii), (iv) or (v);wherein said DNAfragment is expressed when said transgenic plant part is exposed to apest, said transgenic plant part exhibiting increased resistance to saidpest as compared to a non-transgenic plant part of the same part typeand plant species exposed to the same pest under the same conditions,and said increased resistance to said pest being a result of theexpression of said DNA fragment.
 10. A transgenic plant part accordingto claim 9, comprising within its genome A restriction enzyme digestionfragment of genomic grapevine (Vitis) DNA, said fragment encoding aresveratrol synthase, being approximately 4900 base pairs in size andbeing obtainable from plasmid pVSt12t3 by cleavage with EcoRI.
 11. Atransgenic plant part according to claim 9, comprising within its genomea restriction enzyme digestion fragment of genomic grapevine (Vitis)DNA, said fragment encoding a resveratrol synthase, being approximately3400 base pairs in size and being obtainable from plasmid pVSt12t3 bypartial cleavage with EcoRI.
 12. A transgenic plant part according toclaim 9, comprising within its genome a restriction enzyme digestionfragment of genomic grapevine (Vitis) DNA, said fragment encoding aresveratrol synthase, being approximately 12600 base pairs in size andbeing obtainable from plasmid pVSt12t3 by cleavage with SmaI.
 13. Atransgenic plant seed comprising within its genome a DNA fragmentselected from the group consisting of:(i) a restriction enzyme digestionfragment of genomic grapevine (Vitis) DNA, said fragment encoding aresveratrol synthase, being approximately 4900 base pairs in size andbeing obtainable from plasmid pVSt12t3 by cleavage with EcoRI; (ii) arestriction enzyme digestion fragment of genomic grapevine (Vitis) DNA,said fragment encoding a resveratrol synthase, being approximately 3400base pairs in size and being obtainable from plasmid pVSt12t3 by partialcleavage with EcoRI; (iii) a restriction enzyme digestion fragment ofgenomic grapevine (Vitis) DNA, said fragment encoding a resveratrolsynthase, being approximately 12600 base pairs in size and beingobtainable from plasmid pVSt12t3 by cleavage with SmaI; (iv) a cDNAencoding said resveratrol synthase of (i), (ii) or (iii); (v) a DNAencoding said resveratrol synthase of (i), (ii) or (iii); and (vi) asynthetic DNA comprising (i), (ii), (iii), (iv) or (v);wherein said DNAfragment is expressed when said trangenic plant seed is exposed to apest, said transgenic plant seed exhibiting increased resistance to saidpest as compared to a non-transgenic plant seed of the same plantspecies exposed to the same pest under the same conditions, and saidincreased resistance to said pest being a result of the expression ofsaid DNA fragment.
 14. A transgenic plant seed according to claim 13,comprising within its genome a restriction enzyme digestion fragment ofgenomic grapevine (Vitis) DNA, said fragment encoding a resveratrolsynthase, being approximately 4900 base pairs in size and beingobtainable from plasmid pVSt12t3 by cleavage with EcoRI.
 15. Atransgenic plant seed according to claim 13, comprising within itsgenome a restriction enzyme digestion fragment of genomic grapevine(Vitis) DNA, said fragment encoding a resveratrol synthase, beingapproximately 3400 base pairs in size and being obtainable from plasmidpVSt12t3 by partial cleavage with EcoRI.
 16. A transgenic plant seedaccording to claim 13, comprising within its genome a restriction enzymedigestion fragment of genomic grapevine (Vitis) DNA, said fragmentencoding a resveratrol synthase, being approximately 12600 base pairs insize and being obtainable from plasmid pVSt12t3 by cleavage with SmaI.